Cellular localization of membrane-type serine protease 1 and identification of protease-activated receptor-2 and single-chain urokinase-type plasminogen activator as substrates

J Biol Chem. 2000 Aug 25;275(34):26333-42. doi: 10.1074/jbc.M002941200.


Membrane-type serine protease 1 (MT-SP1) was recently cloned, and we now report its biochemical characterization. MT-SP1 is predicted to be a type II transmembrane protein with an extracellular protease domain. This localization was experimentally verified using immunofluorescent microscopy and a cell-surface biotinylation technique. The substrate specificity of MT-SP1 was determined using a positional scanning-synthetic combinatorial library and substrate phage techniques. The preferred cleavage sequences were found to be (P4-(Arg/Lys)P3-(X)P2-(Ser)P1-(Arg)P1'-(Ala)) and (P4-(X)P3-(Arg/Lys)P2-(Ser)P1(Arg) P1'(Ala)), where X is a non-basic amino acid. Protease-activated receptor 2 (PAR2) and single-chain urokinase-type plasminogen activator are proteins that are localized to the extracellular surface and contain the preferred MT-SP1 cleavage sequence. The ability of MT-SP1 to activate PARs was assessed by exposing PAR-expressing Xenopus oocytes to the soluble MT-SP1 protease domain. The latter triggered calcium signaling in PAR2-expressing oocytes at 10 nm but failed to trigger calcium signaling in oocytes expressing PAR1, PAR3, or PAR4 at 100 nm. Single-chain urokinase-type plasminogen activator was activated using catalytic amounts of MT-SP1 (1 nm), but plasminogen was not cleaved under similar conditions. The membrane localization of MT-SP1 and its affinity for these key extracellular substrates suggests a role of the proteolytic activity in regulatory events.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding Sites
  • Cells, Cultured
  • Electrophoresis, Polyacrylamide Gel
  • Endothelium, Vascular / metabolism
  • HeLa Cells
  • Humans
  • Models, Molecular
  • Rabbits
  • Receptor, PAR-2
  • Receptors, Thrombin / metabolism*
  • Serine Endopeptidases / metabolism*
  • Structure-Activity Relationship
  • Substrate Specificity
  • Tumor Cells, Cultured
  • Urokinase-Type Plasminogen Activator / metabolism*
  • Xenopus


  • Receptor, PAR-2
  • Receptors, Thrombin
  • MTSP-1 serine protease
  • Serine Endopeptidases
  • Urokinase-Type Plasminogen Activator